The present invention relates to a method and composition for protecting the central nervous system (CNS) from damage induced by abnormal levels of glutamate, which may result from, for example, a stroke.
The central nervous system is composed of trillions of nerve cells (neurons) that form networks capable of performing exceedingly complex functions.
The amino acid L-glutamic acid (Glutamate), mediates many of the excitatory transactions between neurons in the central nervous system. Under normal conditions, accumulation of glutamate in the extracellular space is prevented by the operation of a recycling mechanism that serves to maintain neuronal glutamate levels despite continual loss through transmitter release (Van der Berg and Garfinkel, 1971; Kennedy et al., 1974). Glutamate, released by glutamatergic neurons, is taken up into glial cells where it is converted into glutamine by the enzyme glutamine synthetase. Glutamine reenters the neurons and is hydrolyzed by glutaminase to form glutamate, thus replenishing the neurotransmitter pool.
This biochemical pathway also serves as an endogenous neuroprotective mechanism, which functions by removing the synaptically released glutamate from the extracellular space and converting it to the nontoxic amino acid glutamine before toxicity occurs. The excitotoxic potential of glutamate (i.e., defined as the ability of excess glutamate to overexcite neurons and cause their death) is held in check as long as the transport process is functioning properly. However, failure or reduction in the transport process such as under ischemic conditions, results in accumulation of glutamate in the extracellular synaptic fluid and excessive stimulation of excitatory receptors, a situation that leads to neuronal death.
Two additional factors complicate and make matters worse: (i) overstimulated neurons begin to release excessive quantities of glutamate at additional synaptic junctions; this causes even more neurons to become overstimulated, drawing them into a neurotoxic cascade that reaches beyond the initial zone of ischemia; and, (ii) overstimulated neurons begin utilizing any available supplies of glucose or oxygen even faster than normal, which leads to accelerated depletion of these limited energy resources and further impairment of the glutamate transport process. This biochemical cascade of induction and progression may continue for hours or days and causes delayed neuronal death.
Abnormally high glutamate (Glutamate) levels in brain interstitial and cerebrospinal fluids are the hallmark of several neurodegenerative conditions. These include acute brain anoxia/ischemia i.e stroke (Graham et al., 1993; Castillo et al., 1996), perinatal brain damage (Hagberg et al., 1993; Johnston, 1997), traumatic brain injury (Baker et al., 1993; Zauner et al., 1996), bacterial meningitis (Spranger et al, 1996), subarachnoid hemorrhage, open heart and aneurysm surgery (Persson et al., 1996; Saveland et al., 1996), hemorrhagic shock (Mongan et al. 1999, 2001), newly diagnosed epilepsy (Kalviainen et al., 1993), acute liver failure (Rose et al. 2000), migraine [Martinez F, Castillo J, Rodriguez J R, Leira R, Noya M, Cephalalgia. 1993 April; 13(2):89-93], stress [Abraham I, Juhasz G, Kekesi K A, Kovacs K J, Stress. 1998 July; 2(3):171-81 and De Cristobal J, Madrigal J L, Lizasoain I, Lorenzo P, Leza J C, Moro M A, Neuroreport. 2002 Feb. 11; 13(2):217-21] and various chronic neurodegenerative diseases such as glaucoma (Dreyer et al., 1996), amyotrophic lateral sclerosis (Rothstein et al., 1990; Shaw et al., 1995), HIV dementia (Ferrarese et al. 2001) and Alzheimer's disease (Pomara et al., 1992).
Thus, one object of medical therapy is to break or eliminate the above described cascade process and thus prevent glutamate associated neuronal damage.
Since glutamate excitotoxicity is mediated by the glutamate receptors, a potential therapeutic approach has been to develop and apply various selective glutamate receptor antagonists in animal models of neurodegeneration. Though displaying powerful neuroprotective effects in experimental stroke and head trauma, the glutamate receptor antagonists failed in clinical trials mainly because of their adverse or even lethal effects (Birmingham, 2002; Lutsep and Clark, 2001; Palmer, 2001).
Attempts have also been made to increase the activity of the various glutamate transporters, present on glia and neurons, which take up Glutamate from the brain interstitial fluid and thereby limit glutamate excitatory action and excitotoxicity. However, none of the above-described approaches have been successful in providing a viable therapeutic approach for lowering glutamate levels.
In light of these failures and the need of alternative approaches to the treatment of neurodegenerative disorders involving glutamate excitotoxicity, the present inventor has hypothesized that excess glutamate in brain interstitial (ISF) and cerebrospinal (CSF) fluids could be eliminated by increasing the relatively poorly studied brain-to-blood glutamate efflux mechanism. Increasing the efflux can be achieved by lowering the glutamate levels in blood thereby increasing glutamate transport from brain ISF/CSF to blood.
While reducing the present invention to practice, the present inventor has uncovered that by maximaly activating two enzymes, glutamate-pyruvate transaminase (GPT) and glutamate-oxaloacetate transaminase (GOT), glutamate degradation in the blood is increased. These two enzymes are two examples of a wider group of enzymes that use glutamate as a substrate in the general formula:
A +GLUTAMATE ←(enzyme)→ C+D whereby A represents the co-substrate, ←(enzyme)→ symbolizes a reversible enzyme and C and D are metabolites of the enzyme. Examples illustrated by this formula include: Glutamate+oxaloacetate ←(GOT)→ 2-keto-glutarate+aspartate, Glutamate+pyruvate ←(GPT)→ 2-keto-glutarate+alanine or Glutamate+4-methyl-2-oxopentoate ←(branched-chain-amino-acid transaminase)→2-ketoglutarate+Valine.
Examples for different substrates that work on the same enzyme include: Glutamate+2-oxohexanedioic acid ←(GOT)→ 2-keto-glutarate+2-aminohexanedioic acid. Glutamate+2-oxo-3-phenylpropionic acid ←(GOT)→2-keto-glutarate+phenylalanine. Glutamate+3-hydroxy-2-oxopropionic acid ←(GOT)→2-keto-glutarate+serine. Glutamate+5-oxopentanoate ←(GPT)→2-keto-glutarate+5-aminopentanoate. Glutamate+4-oxobutanoate ←(GPT)→2-keto-glutarate+4-aminobutanoate. Glutamate+glyoxalate ←(GPT)→2-keto-glutarate+glycine.
Another common feature that these enzymes share is that they use pyridoxal phosphate as a cofactor.
As stated, these enzymes reversibly convert glutamate into 2-keto glutarate. This causes blood glutamate levels to decrease below basal levels thereby creating a far steeper gradient of glutamate levels between the brain ISF/CSF and blood, than normally exists. In order to reach a novel equilibrium, glutamate is transported from the brain to the blood thus lowering the elevated levels of glutamate in the brain. As long as the glutamate levels are low in the blood, this brain-to-blood efflux will continue. In order to keep GOT and GPT working at their maximum levels for the conversion of glutamate into 2-ketoglutarate (Vmax) their respective substrates, oxaloacetate and pyruvate have to be administered at doses at least twice their Km values.
As stated above both glutamate-oxaloacetate transaminase and glutamate-pyruvate transaminase metabolize glutamate, while using oxaloacetate and pyruvate as their respective co-substates. There are however many other transaminases in the body that can metabolize glutamate such as glutamate, branched-chain-amino-acid transaminase, GABA aminotransferases and many others. For each enzyme according to its reaction, a specific substrate such as succinate semialdehyde for 4-aminobutyrate transaminase should be used.
Conversely, although pyruvate and oxaloacetate are possibly the best substrates for the glutamate transaminases, other substrates such as 2-oxohexanedioic acid, 2-oxo-3-sulfopropionate, 2-oxo-3-sulfinopropionic acid, 2-oxo-3-phenylpropionic acid or 3-indole-2-oxopropionic acid instead of oxaloacetate and 5-oxopentanoate, 6-oxo-hexanoate or glyoxalate instead of pyruvate can be used.
The conversion of glutamate to 2-ketoglutarate is reversible. Thus, upon glutamate transformation via an enzymatic reaction into 2-ketoglutarate, there is a buildup of 2-ketoglutarate which can cause the enzyme to work in the reverse direction and convert 2-ketoglutarate into glutamate. It is therefore beneficial to further break down 2-ketoglutarate and in this way insure the continual metabolism of glutamate. One such enzyme that metabolizes 2-ketoglutarate is 2-ketoglutarate dehydrogenase through the general reaction—2-ketoglutarate+lipoamide←(2-ketoglutarate dehydrogenase)→ S-succinyldihydrolipoamide+CO2.
Thus, the present inventor provides a novel approach for protecting neural tissue from damage induced by elevated glutamate levels.